Oral tolerance induction by mucosal administration of cholera toxin B-coupled antigen involves T-cell proliferation in vivo and is not affected by depletion of CD25+ T cells

Abstract

Oral administration of antigens coupled to the B subunit of the cholera toxin (CTB) can dramatically reduce the amount of antigen needed for tolerance induction and has been used in several animal models to suppress conditions where the immune system overreacts to foreign and self-antigens. In this study, the cellular events following oral administration of CTB-coupled antigen was investigated. As a model system, limited numbers of CSFE-labelled cells from influenza haemagglutinin peptide (HApep) T-cell transgenic mice were transferred to wild type mice and the mice were then given CTB-coupled HApep orally. The inductive events of CTB-induced tolerance was characterized by extensive proliferation of HApep-specific T cells in the mesenteric lymph nodes (MLNs) and in the spleen. The proliferating cells up-regulated the gut homing molecule alpha4beta7 and down-regulated the high endothelial venule binding molecule L-selectin. Addition of the whole cholera toxin (CT) to CTB-HApep showed a similar pattern as CTB-HApep feeding, with antigen-specific proliferation in the MLN and spleen and expression of alpha4beta7 on the proliferating cells. However, addition of CT to CTB-HApep, produced a stronger and faster proliferative response and abrogated CTB-HA mediated oral tolerance. Feeding of CTB-HApep expanded CD25+ cells in the MLNs. CTB-induced oral tolerance could, however, not be explained by CD25+ dependent regulatory activity, as oral administration of CTB-HApep to mice depleted of CD25+ cells still gave rise to systemic tolerance. Thus, several mechanisms might co-orchestrate the systemic tolerance seen in response to feeding with CTB-coupled antigen.

Figure 1

Oral administration of CTB-HApep efficiently induces systemic tolerance, which is abrogated by CT. Mice were adoptively transferred with T cells from HA transgenic mice. Four days after the adoptive transfer, mice were fed HA or CTB-HApep. Two days after feeding, the mice were primed in the footpad with 50 µg HApep in emulsified Freund's complete adjuvant. T-cell proliferation in response to HApep was measured on draining PLN cells 2 weeks after priming. (a) Feeding of 15 µg HApep conjugated to CTB three times 2 days apart. Data are means of three separate experiments with a total of nine mice per bar. Results are expressed as mean + SEM. P-value was calculated by one-tailed t-test. (b) Feeding of a single dose of 5 or 30 µg HApep. Dotted bars HApep, hatched bars CTB-HApep. Data shown as means of three mice/group. (c) Mice were fed a single dose of 30 µg CTB-HApep + 5 µg of CT. Data shown are means of three mice/group.

Figure 2

Oral administration of CTB-HApep causes antigen proliferation in vivo. Mice were adoptively transferred with CSFE-labelled T cells from HA transgenic mice. The mice were given antigen orally 2 days after cell transfer. Spleens, MLN and PP were analysed for Vβ8⁺ CSFE labelled cells at various time-points after feeding. (a) Spleen, MLN and PP cells were analysed 4 days after feeding for Vβ8⁺CSFE labelled cells. One representative dot plot is shown. The percentage of CSFE⁺ Vβ8⁺ cells are shown in the upper right corner of each dot plot. (b) means of Vβ8⁺ CSFE^low cells and Vβ8⁻ CSFE^low cells in the indicated organs (two mice/group) are shown. (c) MLN cells were analysed 2 days after feeding. Percentage of CSFE-labelled Vβ8⁺ cells are given in each figure. (d) means of Vβ8⁺ CSFE⁺ cells for two animals/group are shown.

Figure 3

Proliferating T cells in response to CTB-HApep feeding up-regulate α₄β₇ and down-regulate l-selectin in the MLN. CSFE-labelled T cells from HA transgenic animals were adoptively transferred to wild type mice. Two days after transfer, mice were fed antigen. MLN cells were analysed at 2 days after CTB-HApep + CT feeding and four days after CTB-HApep feeding. Cells were gated on Vβ8⁺ lymphocytes and analysed for l-selectin or α4β7.

Figure 4

CTB-HApep feeding expands CD25⁺ cells in vivo. CSFE-labelled T cells from HA transgenic animals were adoptively transferred to wild type mice. Two days after transfer, mice were fed antigen. MLN cells were analysed at 4 days after feeding. Cells were gated on Vβ8⁺ lymphocytes and analysed for CD25. (a) One representative plot is shown. (b) Analyses of proliferating CD25⁺ cells in response to feeding (two mice/group) are shown.

Figure 5

CTB-induced tolerance is intact in the absence of CD25⁺ cells. Mice were adoptively transferred with T cells from HApep transgenic mice. In the upper panel, mice injected with the CD25 depleting antibody PC61 or control mice were fed with CTB-HApep. Four days later, the mice were fed CTB-HApep and primed later in the footpads with HA. Proliferative responses against HA was measured 2 weeks after priming. In the lower panel, a representative plot for CD25 expression by blood lymphocytes 18 days after PC61 antibody injection is shown. R2 represents all CD25⁺ cells, R3 represents CD25^bright cells.

Figure 6

Adoptive transfer of CD25⁻ cells leads to oral tolerance in response to CTB-HApep feeding. T cells from HA transgenic mice were depleted of CD25⁺ cells and adoptively transferred to wild type mice. The mice were then fed CTB-HApep and primed in the foot-pads with HApep. HApep-specific proliferative responses of draining PLN cells was measured 2 weeks after priming.